The major objective of this research project is to investigate the pathophysiological significance of BIL-ALB, recently confirmed by us to be a significant fraction of serum conjugated bilirubin (BR) that is apparently covalently bound to albumin is sera from patients with hepatobiliary diseases. Specific aims: To investigate the chemical nature and site of formation of BIL-ALB and to determine if other organic anions also bind covalently to albumin in the presence of cholestasis. Methods: BIL-ALB and other BR fractions will be measured by a reverse-phase HPLC method. BIL-ALB will be purified from pooled, jaundiced sera by sequential column chromatography. The identity of the conjugated BR species will be determined by thin-layer chromatography (TLC) of ethyl anthranilate azoderivatives and products of methanolysis with sodium methoxide. Proteolysis of BIL-ALB followed by purification and Edman-degradation of BR-tagged 'chromopeptides' will be used to identify the amino acid site/s of binding. Chromic acid oxidation and TLC separation of the liberated imides would aid in identifying the nature of the BR-albumin bond. An isolated perfused rat liver model will be used, along with detailed kinetic studies of any non-enzymatic formation of BIL-ALB in in vitro systems to determine the site of BIL-ALB synthesis- in the liver or in peripheral circulation. Any alteration in the turnover rate of albumin resulting from BIL-ALB formation will be studied. In addition, covalent binding of estrogen and benzodiazepene metabolites to albumin will be looked for in patients with cholestasis. Significance: These studies should better our understanding of the metabolism, transport and protein-binding of BR and other organic anions in the presence of hepatobiliary diseases and would lead to a more complete study of protein binding of organic anions. After completing 2 years of laboratory research supported by an NIH Institutional Training grant in Hepatology, the candidate was appointed to the Digestive Diseases Division faculty in July, 1983. The Yale Liver Study Unit is an ideal environment for this project. The co-sponsors are well experienced researchers in hepatocellular function and metabolism. Dr. E. Gordon is an internationally renowned expert in bilirubin chemistry and metabolism. Thus, both equipment and technical expertise are excellent. Interaction with basic science department such as Biochemistry and Chemistry is readily available